Eosinophil cationic protein (ECP), a member of the ribonuclease A (RNase A) superfamily, is found in the specific granules of eosinophilic leukocytes. It is a single polypeptide with a molecular mass ranging from 16 to 21.4 kDa due to varying degrees of glycosylation. It shows a 67% amino acid sequence identity with eosinophil-derived neurotoxin (EDN), another eosinophil-secreted RNase. Although ECP shares the overall three-dimensional structure of RNase A, it has relatively lower RNase activity (Boix, E., et al. (1999) Journal of Biological Chemistry 274, 15605-15614). ECP released by activated eosinophils contributes to the toxicity against helminth parasites, bacteria, and single-strand RNA viruses (Lehrer, R., et al. (1989) Journal of Immunology 142, 4428-4434; Domachowske, J. B., et al. (1998) Nucleic acids research 26, 3358-3363). Together with other proteins secreted from eosinophils such as EDN, eosinophil peroxidase (EPO; also EPX), and major basic protein (MBP), ECP is thought to cause damage to epithelial cells, a common feature of airway inflammation in asthma (Gleich, G J. (2000) Journal of Allergy and Clinical Immunology 105, 651-663).
The mechanism underlying the cytotoxic property of ECP is unclear. It has been hypothesized that ECP cytotoxicity is due to destabilization of lipid membranes of target cells (Young, J., et al. (1986) Nature 321, 613-616), and the degree of cytotoxicity is dependent on the cellular concentration (Carreras, E., et al. (2005) Molecular and Cellular Biochemistry 272, 1-7). The binding of ECP to target cells has been attributed to its high arginine content (estimated pI=10.8), which facilitates the interaction between ECP and negatively charged molecules on the cell surface (Carreras, E., et al. (2005) Molecular and Cellular Biochemistry 272, 1-7; Carreras, E., et al. (2003) Biochemistry 42, 6636-6644). Recently, we found that binding and endocytosis of ECP into bronchial epithelial cells were greatly dependent on the cell surface glycosaminoglycan (GAG), specifically heparan sulfate proteoglycans (HSPG) (Fan, T. C., et al. (2007) Traffic 8, 1778-1795). The cytotoxicity of ECP was severely reduced toward cell lines with heparan sulfate (HS) deficiency.
Heparin and HS are complex polysaccharides composed of alternating units of hexuronic acid and glucosamine. The uronic acid residues of heparin typically consist of 90% L-idopyranosyluronic acid and 10% D-glucopyranosyluronic acid (Capila, I. and Linhardt, R. J. (2002) Angewandte Chemie International Edition 41, 391-412). The N position of glucosamine may be substituted with an acetyl or sulfate group. The 3-0 and 6-0 positions of glucosamine and the 2-0 of uronic acid may be sulfated. Through the combination of different negatively charged moieties, heparin and HS have been demonstrated to bind a variety of proteins with diverse functions, including growth factors, thrombin, chemokines and viral proteins. The HS chains contain domains with a high level of sulfation and epimerization (S-domains), regions with mixed N-acetylation and N-sulfation (NA/S-domains), and unmodified domains that are mostly N-acetylated and contain little sulfate (Tumova, S., et al. (2000) The international journal of biochemistry & cell biology 32, 269-288). Because HS chains contain heparin regions, heparin and its mimetics can be used to study interactions between proteins and polysaccharides.
The structure of ECP has been determined and refined to a resolution up to 1.75 Å, displaying a folding topology that involves three α helices and five β strands (Mallorqui-Fernandez, G, et al. (2000) Journal of Molecular Biology 300, 1297-1307). The most interesting feature is the 19 surface-oriented arginine residues, conferring a strong basic character to ECP. However, the heparin binding site in ECP has not been identified. Heparin binding domains within proteins usually contain a high proportion of positively charged residues, which bind to the acidic groups of heparin through electrostatic interactions. It has been proposed that the three-dimensional structure of the HS chain is critical for protein binding (Hileman, R. E., et al. (1998) BioEssays 20, 156-167). However, not much is known about the three-dimensional structure of HS. After examining a series of heparin-binding protein sequences, Cardin and Weintraub proposed that the pattern XBBBXXBX or XBBXBX (where X represents hydrophobic or uncharged amino acids, and B represents basic amino acids) is responsible for HS binding to other proteins (Cardin, A. D. and Weintraub, H. J. (1989) Arteriosclerosis (Dallas, Tex. 9, 21-32). In addition, the following sequences have also been reported to serve as heparin binding motifs.
BBXXBBBXXBB (where B is a positively charge residue (arginine, lysine or hystidine) and X is any residue) (Olenina, L. V, et al. (2005) J Viral Hepat 12, 584-593).
BXXBBXB (where B is a basic residue and X is any residue) (Wu, H. F., et al. (1995) Blood 85, 421-428).
XBBBXXBBBXXBBX (where B is a basic residue and X is any residue) (Andersson, E., et al. (2004) Eur J Biochem 271, 1219-1226; Sobel, M., et al. (1992) The Journal of biological chemistry 267, 8857-8862).
TXXBXXTBXXXTBB (where B is a basic residue, X is any residue, and T defines a turn) (Capila, I. and Linhardt, R. J. (2002) Angewandte Chemie International Edition 41, 391-412; Hileman, R. E., et al. (1998) BioEssays 20, 156-167).